Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide (1) and is the major indication for liver transplantation in the United States (2, 3). While therapy with PEGylated Interferon-alpha (PEG-IFN) plus ribavirin (RBV) provides sustained virologic response (SVR) in 40-50% of patients overall, the SVR rate is only about 20% in African Americans and Hispanics, who have a higher frequency of single nucleotide polymorphisms associated with poor response (4, 5). Addition of direct-acting antivirals (DAAs) targeting the HCV NS3/4A protease increases SVR rates to 68-75% in previously untreated patients, but such regimens still suffer from the limitations of PEG-IFN/RBV. Thus, drug developers are seeking combinations of DAAs that will reduce or eliminate the need for PEG-IFN and/or RBV while providing an adequate barrier to resistance. It was recently reported that a small number of genotype 1a and 1b patients achieved SVR following treatment with a combination of two DAAs targeting NS3/4A and NS5A. While these results provide proof of concept for SVR without use of PEG-IFN/RBV, biological data to inform the selection of such DAA combinations are limited. The preponderance of preclinical characterization of DAAs and combinations has been obtained using subgenomic HCV replicons in Huh-7 hepatoma-derived cell lines, a recombinant system that omits important features of the viral life cycle. Systems have been developed that recapitulate the complete HCV life cycle in hepatoma lines and primary human hepatocytes/hepatoblasts. This study proposes to interrogate the effects of DAAs in cell systems that support the complete HCV life cycle. These studies will deepen our understanding of key biological processes involved in viral replication and provide new insights into DAA mechanisms of action that will be translatable to clinical studies. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major public health burden, with approximately 130 million people infected worldwide, and HCV-associated liver disease is the main indication for liver transplant in the United States. We propose to characterize direct-acting antiviral agents and identify combinations with the greatest ability to eliminate infection by usin state of the art analytical methods and cell culture systems that reproduce the complete HCV life cycle in primary human hepatocytes.